Endogenous daily (circadian) rhythms govern many behavioral and physiological functions. A master pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN) controls these oscillations, but the transcriptional-translational feedback loops that generate them are distributed in cells throughout the body. These peripheral circadian oscillations persist for many cycles, if not indefinitely, in isolated organs after they are placed in culture. The SCN generates neural and endocrine signals in order to regulate circadian oscillations in peripheral tissues, but the way in which these signals act as entraining cues (zeitgebers) and the determinants of the phase taken by peripheral oscillators to the master pacemaker are poorly understood. Although powerful tools are available in the form of transgenic mice, novel approaches are required to determine the behavior, the physiological role, and the importance of the peripheral oscillators. The experiments described in this R21 application will utilize a mouse luciferase reporter construct in order to establish whether the SCN entrains hepatic oscillations, and to determine which specific signals serve as zeitgebers. The roles of the neurotransmitters epinephrine and acetylcholine, as well as hormones including glucocorticoids, insulin and glucagon, will be examined. The influence of fluctuations in glucose concentration will also be tested. A novel-flow through system will be used in which physiological signals (hormones or neurotransmitters) are added to organ culture chambers containing replicate sections of liver taken from transgenic Per2::luc mice. The ability of these signals to regulate period and phase will be established by luminometry, and we will determine whether the criteria of entrainment are met. For the first time for a peripheral oscillator, the range of entrainment will be examined and phase response curves wil be constructed. The health relevance of circadian organization will be evaluated as the impact of entrainment of the hepatic clock to various periods upon secretion of VLDL-cholesterol is determined through assay of the perifusion effluent. Once this luminometry system is established, it may be used to analyze not only entrainment of other hepatic circadian rhythms, but also the clock-like behavior of other organs. These studies will provide important new tools for analysis of the multi-oscillator system that governs normal physiological function and whose malfunction may contribute to disease. [unreadable] [unreadable] [unreadable]